The generation of amyloid beta-protein (Abeta) by proteolytic cleavage of the amyloid precursor protein (APP) is a central pathogenic event in Alzheimer's disease. The cleavage event that generates the carboxyl-terminus of Abeta is mediated by an unknown "gamma-secretase". Presenilin-1 (PS1) mutations that cause familial Alzheimer's disease alter the gamma-secretase cleavage of APP. We and others have recently shown that PS1 coordinately regulates Abeta gamma-secretase cleavage and a similar cleavage of Notch-1. This proteolytic cleavage of Notch- 1 releases the intracellular domain, a critical step in the activation of the Notch-1 signaling pathway. The overall goal of this proposal is to elucidate the mechanism by which PS1 regulates APP and Notch-1 proteolytic cleavage, and to characterize the proteases involved. Our preliminary studies demonstrate that PS1 promotes proteolytic cleavage of Notch-1, leading to nuclear translocation of the cleaved fragment and transcriptional activation. Our findings further suggest that PS1 may promote the trafficking of APP and Notch-1 to cellular sites where gamma-secretase cleavage occurs. Furthermore, our preliminary studies provide evidence that calpain I may be the gamma-secretase that produces Abeta40 and generates the Notch-1 intracellular fragment. A different gamma-secretase appears to mediate the cleavage that gives rise to the 42-amino acid form of Abeta (Abeta42). The studies in this proposal will investigate the role of calpain I as a gamma-secretase which cleaves APP and Notch-1, and the regulation of gamma-secretase activity by calcium. The mechanism of action of PS1 in the regulation of gamma-secretase activity and the effect of pathogenic PS1 mutations will also be investigated. These studies will utilize cortical and fibroblast cultures from PS1-knockout and wild-type mice, as well as stably transfected cell lines that express pathogenic PS1 mutations. Three potential mechanisms will be explored: 1) Whether PS1 promotes proteolytic cleavage secondary to an effect on protein trafficking; 2) Whether PS1 is a co- factor which activates gamma-secretase; and 3) Whether PS1 is the gamma-secretase. These issues are central to understanding the pathogenesis of Alzheimer's disease and may provide new directions for therapeutic intervention.